Local calcium signal transmission in mycelial network exhibits decentralized stress responses

Abstract Many fungi live as mycelia, which are networks of hyphae. Mycelial networks are suited for the widespread distribution of nutrients and water. The logistical capabilities are critical for the extension of fungal survival areas, nutrient cycling in ecosystems, mycorrhizal symbioses, and virulence. In addition, signal transduction in mycelial networks is predicted to be vital for mycelial function and robustness. A lot of cell biological studies have elucidated protein and membrane trafficking and signal transduction in fungal hyphae; however, there are no reports visualizing signal transduction in mycelia. This paper, by using the fluorescent Ca2+ biosensor, visualized for the first time how calcium signaling is conducted inside the mycelial network in response to localized stimuli in the model fungus Aspergillus nidulans. The wavy propagation of the calcium signal inside the mycelium or the signal blinking in the hyphae varies depending on the type of stress and proximity to the stress. The signals, however, only extended around 1,500 μm, suggesting that the mycelium has a localized response. The mycelium showed growth delay only in the stressed areas. Local stress caused arrest and resumption of mycelial growth through reorganization of the actin cytoskeleton and membrane trafficking. To elucidate the downstream of calcium signaling, calmodulin, and calmodulin-dependent protein kinases, the principal intracellular Ca2+ receptors were immunoprecipitated and their downstream targets were identified by mass spectrometry analyses. Our data provide evidence that the mycelial network, which lacks a brain or nervous system, exhibits decentralized response through locally activated calcium signaling in response to local stress.


Introduction
Most fungi form mycelia by repeatedly elongating and branching hyphae (1,2). They secrete numerous degradative enzymes outside, degrade biopolymers in the environment, and get nutrition by absorbing them during mycelial development. Mycelia are well-suited for adsorption on nutritional substrate and entrance into them, which adds a great advantage to fungi in infection/ decomposition of large and solid substrates (2)(3)(4). The network structure of mycelium allows for the widespread distribution of absorbed nutrients across the network. This logistical role is critical for the extension of fungal survival area, nutrient cycling in ecosystems, mycorrhizal symbioses, and virulence (5)(6)(7).
Mycorrhizal networks connect neighboring plants in the soil and contribute to their nutritional distribution (8). In addition to nutrition logistics, signal transmission such as stress response is predicted to be crucial for the mycelial function and robustness (9), although little is known about signal transduction in mycelial networks.
Cells of multicellular organisms are integrated into a single living system through cellular communication. Calcium signaling, defined as a change in intracellular Ca 2+ concentration, serves as a second messenger for transmitting biological information between cells in several species (10). Calcium signaling, for example, is required for chemical exchange and information transfer between neurons in neural networks (11). In another example, when plants are exposed to environmental stressors, such as drought or damage, calcium signaling is activated, which triggers downstream responses (12). When one leaf is damaged, calcium signal moves via the veins, which carry water and nutrients throughout the plant, to leaves away from the injured leaf and throughout the plant body (13). Calmodulin (CaM) and calcium/ CaM-dependent protein kinase (CaMK) are the principal intracellular Ca 2+ receptors and are responsible for conveying these signals to numerous target proteins (14). Ca 2+ -bound and activated CaM binds directly to target proteins, altering their activity. CaMK phosphorylates target proteins to regulate their activity.
Most Dikarya fungi form multicellular mycelia whose hyphae are segregated by septa, although the cytoplasm is continually linked because the septa have tiny pores (15). Recent research has shown that oscillatory Ca 2+ inflow at a hyphal tip influences the timing of actin depolymerization and exocytosis, resulting in oscillatory hyphal growth (16,17). This study investigated the conduction of calcium signaling within the mycelial network and each hypha in response to localized stimuli. Green fluorescent protein (GFP)-trap of CaM/CaMK and mass spectrometry analyses identified downstream targets of calcium signaling.

Conduction of calcium signaling in mycelium by cutting stress
Intracellular Ca 2+ content was visualized by red fluorescent Ca 2+ biosensor, R-GECO, in the model fungus A. nidulans (16). The red fluorescence in the mycelium was monitored by a wide-field fluorescence microscope, when the colony, ∼10 mm diameter after 2 days incubation, was cut with a razor blade (13) (see Methods). We cut the mycelial edge was along the parallel direction to the hyphal growth to investigate how the signal spreads horizontally in mycelium to other adjacent hyphae (Fig. 1A, Video S1). Calcium signal appeared simultaneously at ∼500 μm from the cut site, peaked at 10 s (Figs. 1B and S1A, t = 33), and gradually faded until 120 s (Fig. 1C, D). The initial fluorescence was stronger near the cut (>300 μm), diminished as the distance increased from 300 to 600 μm, and showed no significant difference further than 600 μm (Fig. 1A-D). The signal was stable throughout the hyphae around the cleft (>300 μm). At 300-600 μm from the cut site, following the initial fluorescence's faded, the signal often appeared, spread, and disappeared within the hyphae (Video S1). Initial fluorescence was low and quickly disappeared in mycelia far from the cut (<600 μm). The longest signal was detected at distance of ∼1,000 μm from the cut site (Fig. 1D).
Following the initial fluorescence's faded, short-lived signals were observed at the limited area (<50 μm) of tips or in the middle of hyphae at various locations in the mycelium (Video S1). The Ca 2 + signal appeared to propagate between different hyphae in close and spread within the mycelium (Figs. 1E and S1B). The signal intensity decreased as the distance from the cut site increased. There appears to be a correlation between signal appearance time and distance from the cleavage site (Fig. S1A). The same hyphae rarely blinked repeatedly.
When the similar size mycelium was cut around the edge vertically to the hyphal growth direction (Fig. 1F, G, Video S2), calcium signal immediately emerged throughout the mycelium near the cleavage site (<500 μm). The signal intensity decreased gradually as further away from 500 μm (Fig. S1C), which is like Fig. 1B. Around 1,000 μm from the cut site, a signal peak appeared in the middle of hyphae (Fig. 1G, H). This suggests that the diffuse signal from the cleavage extends to around 1,000 μm. The signal decreased drastically at further distance (Fig. S1D). In the box area 800-1,400 μm from the cut site, there were two frequent signal appearances, suggesting signal propagation by the first and second waves whose transfer rates were 41 and 50 μm/s, respectively ( Fig. 1I-L).
The smaller colony, ∼5 mm diameter after 1 day incubation, was cut in half (Fig. S1E, F). Calcium signal immediately emerged throughout the mycelium near the cleavage site and weaker away from the cleavage. The signal appeared in the hyphae <700 μm from the cleavage. When the 2-day-cultured mycelia were cut at Fig. 1A and F, the signal diffused over 1,000 μm regardless of the cutting way, but when the younger mycelium was cut, the signal only traveled 700 μm (Fig. 1H). When 7-or 14-daycultured mature mycelia were cut around the edge vertically, the signal diffused around 1,500 μm from the cleavage (Fig. 1H, M, Video S3). These results suggest that the distance of signal transmission depends on the maturity of the mycelial network and that calcium signals travel only 1,500 μm maximum in the mycelial network. The fluorescence intensity near the cleavage was comparable in 2-, 7-, and 14-day colonies and higher than that of 1 day colony (Fig. S1G). The analysis of effect by the cleavage on mycelial growth found mycelial elongation was delayed only in the area severed near hyphal tips, otherwise most were unaffected (Fig. 1N, O, Video S4).

Conduction of calcium signaling in mycelium by drop of EtOH or NaCl
When 1 μL ethanol was dropped to the edge of mycelium as the dehydration stress, whose diameter is ∼10 mm, calcium signal appeared simultaneously in the dropped area, whose intensity reached its peak after 15 s, decreased slowly until 160 s ( Fig. 2A, B, Video S5). The fluorescence hardly spread out the dropped area (Figs. 2C, D and S2A). The calcium signal blinked repeatedly at the tips of many hyphae. Some hypha blinked 5 times every 35 s for 175 s ( Fig. 2E-G, gray), other blinked a quick weak signal 4 times in 30 s (orange t = 63-99). The maximum value of fluorescent intensity that blinked multiple times gradually decreased with time (Fig. 2F). Outside of the ethanol dropped area, hyphae rarely blinked, and the number of times they blinked was limited to two or three (Fig. S2B). When 1 μL 3 M NaCl was dropped to the edge of mycelium as the salt stress, whose diameter is ∼10 mm, a few hyphal tips fluoresced in the dropped area and faded quickly (Fig. 2H, I, Video S6). After 80 s period of non-fluorescence, the fluorescent intensity of many hyphae gradually increased ( Fig. 2H-K). The calcium signal spread from the middle of hyphae to both the tip and the base at a rate of 4.9 ± 1.8 μm/s (Figs. 2L and S2D). After 150 s, most of the hypha in the NaCl-dropped area showed stable fluorescence that was diffuse throughout the hyphae (Fig. 2J, K). The signal intensity gradually decreased until 300 s (Fig. S2G). Outside of the dropped area, a small number of hyphae briefly blinked one or two times (Fig. S2F). Similar signal patterns were observed in the 7-day-cultured colony in response to ethanol and NaCl (Fig. S2H, Video S7).
Comparing the time course of the stress responses of cleavage, ethanol, and NaCl, the cleavage reached its maximum value immediately (<5 s), while the ethanol stress reached its peak after a slight delay of 15 s. The cleavage showed a higher maximum  than the ethanol or NaCl treatment (Fig. 2M). The NaCl stress showed an increase in signal after 90 s, reaching a peak comparable with the ethanol treatment. Mycelial elongation was delayed only in the area dropped EtOH or NaCl, otherwise most were unaffected (Fig. S2I, J, Video S3).

Calcium signal conduction through hyphae
Calcium signal was analyzed in each hypha stimulated by point laser irradiation (see Methods). When the point laser was irradiated on the hyphal tip, calcium signals quickly appeared repeatedly, each signal spreading backward and disappearing (Fig. 3A, B, Video S8). Within 2 min, the signal appeared 4 ± 2 times (Fig. 3C). The kymograph analysis yielded the conduction time, distance, and velocity: 11 ± 5 s, 25 ± 15 μm, and 4 ± 2 μm/s (Fig. 3D), respectively. The mean interval time of calcium signals was 18 ± 12 s (n = 48 signal). As observed in Fig. 2L, calcium signal sometimes diffused in both directions at the middle of the hyphae, (Fig. 3E, Video S9). There was no significant change in conduction time and velocity between the tip and the middle of hyphae (Fig. 3D). The calcium signal pass through the septa, but it slowed once there (Fig. 3F, Video S9). Flickering calcium signals were transmitted to neighboring hyphae, but signal intensity and number of flickers decreased (Fig. 3G).
The number of calcium pulses and intervals were monitored when the laser irradiation time was changed to 5 and 20 s instead of 10 s. The average calcium pulses numbers were 3 ± 2, and 5 ± 2, respectively (Fig. 3C). The average interval times were 21 ± 17 and 15 ± 9 s, respectively (Fig. 3C). As laser irradiation time increased, the number of calcium pulses increased, and the interval became shorter. Without laser stimulation, the average interval between pulses was 26 ± 7 s (16).
The laser irradiation stopped the hyphal growth for a short time. A new polar site emerged from the tip after several minutes, and tip growth began. Laser irradiation caused F-actin and secretory vesicles at the apex of the mycelial tip to lose their signal (Figs. 3H and S3A, Video S10). When hyphal growth resumed, F-actin and secretory vesicles localized to the new polar site. The results suggest that calcium signal elicited by laser irradiation have a role in quick actin depolymerization and membrane transport pausing. In general, intracellular Ca 2+ levels directly regulate actin assembly and vesicle fusion (18,19). Conversely, when actin polymerization or membrane transport was inhibited by chemical treatment, hyphal growth ceased, and no periodic calcium influx was also observed (Figs. 3I and S3B, C).

Target proteins of CaM and CaMKs
Although calcium signal plays a role in the rearrangement of the actin cytoskeleton and vesicle transport in hyphae, little is known about how calcium signal controls these processes. The A. nidulans genome possesses genes encoding sole calmodulin (caM) and three CaMKs (cmkA, cmkB, and cmkC) (Fig. S4A). caM, cmkA, and cmkB are essential since they are required for progression of mitosis (20)(21)(22). cmkC is not essential, but the spore germination and nuclear division are delayed, the colony is sensitive to osmotic stress (22,23).
CaM-mRFP localized at the apex of growing hyphae and at the spindle pole body (Fig. 4A) (24,25). Our movie shows CaM moved to the hyphal apex as microtubules elongated (Fig. S4B, Video S11). CmkA, CmkB, and CmkC, each with respective GFP tags and expressed under the native promoter, are present in the cytoplasm (Fig. 4A). These strains show no severe phenotype, indicating CaM-mRFP and CaMK-GFP fusion proteins are functional. qRT-PCR analysis did not detect any changes in the expression levels of CaMKs in response to osmotic stress and treatment with inhibitors of actin polymerization, membrane transport, or calcium channel (Fig. S4C). The apical localization of CaM-mRFP disappeared in response to EtOH or NaCl treatment (Fig. S4D).
CaM-and CaMK-interacting proteins were immunoprecipitated by using RFP-trap or GFP-trap and identified by LC-MS/MS (see Methods). SDS-PAGE confirmed that the bands of these proteins correspond to those of CaM and CaMKs (Fig. S4E). In two independent experiments, we focused on proteins having LC-MS/ MS intensities that were 10-fold higher than the negative control (wild type), higher than 3.E + 06, and sequence coverages of >10% (Fig. 4B, Table S1). In the case of CmkB, proteins were focused with an intensity more than 4.E + 07 and sequence coverages >10% since many mitochondrial proteins were detected. CaM, CmkA, CmkB, and CmkC each have 11, 16, 44, and 7 potential target proteins, respectively. There was little overlap between target proteins (Fig. 4C), implying that signaling to distinct targets is functionally differentiated.
As CaM-mRFP-interacting proteins, we identified three CaMKs and calcineurin, a calmodulin-dependent phosphatase, subunits A and B (Fig. 4B). In addition, endocytosis and exocytosis motor proteins (myosin-1, myosin-5, and kinesin-3) (26)(27)(28) and an ortholog of the vacuolar sorting-associated protein Vps13 (29) were discovered. These proteins did not coprecipitate with CaM-mRFP when EDTA was added to the cell lysate, demonstrating that their interaction with CaM is Ca 2+ dependent. GO and enrichment analysis (cluster frequency/background frequency) showed that proteins involved in vesicle trafficking and nuclear division regulation were enriched in the CaM-interacting proteins (Table S2).

Discussion
Intracellular Ca 2+ dynamics have been shown in the hyphae of several filamentous fungi (16,38,39). This study is the first to show how calcium signals propagate within the mycelial network. Localized calcium signal was revealed when a piece of the mycelium was cut, and it spread outward for around 1,500 μm. Dropping ethanol or NaCl on a piece of the mycelium also activated the signal in the area where it was dropped. Mycelia lack a brain or nervous system, therefore the control is decentralized rather than centralized (2). Even if stress is perceived in one area of the mycelium, there is no need to alert the entire mycelium, rather, it is more efficient to respond to stress at that site. In fact,  the growth of the colony was slowed only in the stressed areas, while most of the rest of the colony continued to grow without delay. The mycelium extending in all directions and making decisions at each location would be suitable for mycelial growth and adaptation.
Ca 2+ oscillations are a ubiquitous means of signaling in all cells. Positive feedback, or in combination with negative feedback, can trigger the Ca 2+ oscillations that arise from Ca 2+ -induced Ca 2+ release (40). The shape of oscillations, which is characterized by their amplitude and phase, can efficiently transmit different cellular responses in both plant and mammalian cells (41). Our results indicate the shape of oscillations varies depending on the kinds of stress and proximity to the stress. As pointed laser irradiation time increased, the number of Ca 2+ influxes increased, and the interval became shorter. The degree of the stress or the distance from the stress can activate differentially the downstream pathways, although deciphering that message requires further analysis.
In response to laser irradiation, actin cytoskeleton depolymerizes at the hyphal tip, which also stops vesicular transport and hyphal extension. After a short time, cell polarity and actin cytoskeleton are reconstituted, which resume vesicular transport and hyphal extension. Motor proteins (myosin-1, myosin-5, and kinesin-3) were identified as targets of CaM. The former is involved in actin organization, while the latter are involved in vesicle transport. CaM is localized at the hyphal tips, where actin polymerization and exocytosis occur, and at SPBs where the minus ends of the microtubule bind. CaM translocation to the hyphal tip is associated with microtubule elongation, which is expected to function in membrane transport in association with kinesin-3 and myosin-5. CmkA is also involved in membrane traffic through interaction with ArlA (ARF). ChsB, a class III chitin synthase, is transported by vesicle to hyphal tips and plays an essential role in hyphal tip growth (37,42), which is co-precipitated with CmkC. Chitin synthase, Chs3, of Candida albicans is phosphorylated, and both phosphorylation and dephosphorylation are required for its correct localization and function (43). CmkC may be a link between calcium signaling and cell wall synthesis by phosphorylating the chitin synthase. Calcineurin is a target of the immunosuppressive drugs and is important in fungal virulence and drug resistance (44). Transcription factor Crz1, one of calcineurin targets, is involved in the regulation of stress response, cell wall integrity, and drug resistance through dephosphorylation and nuclear translocation (45). The cell wall integrity signaling pathway may also be involved in the stress response; however, since there is no overlap with that pathway other than ChsB (46), the functions are expected to be distinct from the calcium signaling pathway. Notably, a putative transcription factor is identified here as CmkA-associated proteins.
We have visualized the calcium oscillations in single hypha and have found a link to actin and exocytosis (16). We searched for targets of CaM and CamKs to reveal what molecules downstream of the calcium signal are involved in regulating hyphal growth. On the other hand, even if the response of a single hypha alone is clarified, since the response of overall mycelial network is unknown, we investigated how calcium signals are transmitted in the mycelial network. By exploring upstream and downstream of previous findings, respectively, this paper links phenomena at different hierarchies from macro mycelia to single hyphal cell, and to target proteins. Our results show that local calcium signal transmission in mycelial networks exhibits decentralized stress responses.
The mycelial network of Ascomycota A. nidulans was analyzed in this study. Given the differences in the fundamental structure between arbuscular mycorrhizal networks and Basidiomycota mycelial networks, we must be careful when determining if this finding applies to both. AM fungi optimize the mycelial networks by adjusting transport and distribution of nutrients during growth, so that flow is concentrated in the major hyphae, which become thicker and have thicker cell walls, whereas unnecessary hyphae become empty and are separated by septa (47). Ectomycorrhizal and saprotrophic basidiomycetes form mycelial strands, which are aggregates of multiple hyphae, and some form rhizomorphs with differentiated thick-walled fiber hyphae and vessel hyphae, with diameters of ∼10 to 15 μm, that are likely to improve flow (2,48). It is still difficult to visualize the fluidity of mycelial networks, although there is growing evidence that quantum dots and fluorescent proteins can move within mycelial networks (49). Attempts are being made to predict the ecological importance of mycelial networks by modeling their development and features (50). Optimization of logistics in mycelial networks appears to be related to fungal memory (51). Electrical signaling in mycelial networks may also be involved in intelligent communication in fungi (52). There is a wide and unexplored field of research behind the study of the transmission of materials and information in mycelial networks, which reveals fundamental properties of fungi.

Fungal strains and media
The strains of filamentous fungi used in this study are listed in Table S3. Tagging with GFP is described in Supplemental Methods and Table S4.

Point laser irradiation
Aspergillus nidulans cells were grown on minimal medium agar plates at 30°C for 2 days. Then, the edges of colonies were cut into 5 mm squares and placed in glass dishes for observation under an inverted microscope IX-83 (Olympus). A localized heat shock was imposed on individual hypha using an IR-LEGO 1000 system (Sigma Koki) (54,55), equipped with custom-made UPlanSApo 20x/0.75 and UPlanSApo 40x/0.95 objective lens (Olympus), operating at 15 mW for 5-20 s. MetaMorph and Image J software were used for image analysis.

GFP-trap and protein identification by Lc-MS/Ms
All information is shown in Supplemental information. The proteomic data (Table S1) have been deposited in the ProteomeXchange Consortium via the jPOSTrepo with the data set identifier PXD027777 for ProteomeXchange and JPST001285 for jPOSTrepo.
Other methods are shown in supplemental information.